Bioactive Eudesmane sesquiterpenes from Artabotrys hongkongensis Hance

Abstract A new naturally occurring trinoreudesmane sesquiterpene, artahongkongol A (1), together with seven known eudesmane sesquiterpenes (2−8), was isolated from the stems and leaves of Artabotrys hongkongensis Hance. Among them, 1 is a rare trinoreudesmane sesquiterpene containing 12 carbon atoms on the carbon skeleton. All known compounds (2−8) were isolated from the genus Artabotrys for the first time. The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons with data reported in the literature. All isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480 in vitro. Compounds 1−8 showed significant inhibitory effects against various human cancer cell lines with IC50 values ranging from 0.57 to 15.68 μM. Graphical Abstract


Introduction
The genus Artabotrys (Annonaceae) comprising about 100 species is mainly distributed in tropical and subtropical regions. There are about four species of this genus in China, namely, A. hongkongensis Hance, A. pilosus Merr. et Chun, A. hexapetalus (L. f.) Bhandari and A. hainanensis R. E. Fries. Among them, A. hongkongensis Hance grows widely throughout the southern part of China (Editorial Committee of Flora of China 1979). Previous phytochemical investigations on this genus had resulted in the isolation of various types of compounds including alkaloids, sesquiterpenes, cyclohexenes, steroids, long chain hydrocarbons, and flavonoid glycosides, which exhibited a broad range of biological activities, such as anti-tumor, anti-malarial, anti-inflammatory, anti-bacterial, and anti-fertility activities (Wong and Brown 2002;Somanawat et al. 2012;Nyandoro et al. 2013;Zhou et al. 2015;Liu et al. 2015;Tan et al. 2015;Xi et al. 2017;Tang et al. 2018). As a Chinese endemic medicinal plant, up to now, there is only a preliminary investigation on the chemical composition of A. hongkongensis Hance performed by us previously (Wu et al., 2017;Liu, Tang, et al. 2018). Our preliminary experimental results showed that the 90% EtOH extract of the stems and leaves of A. hongkongensis Hance exhibited significant cell growth inhibitory activities against various human cancer cell lines with IC 50 in the range of 3.48-22.39 lg/mL in vitro. As a part of our ongoing research into structurally and biologically interesting natural products from medicinal plants in China (Fu et al. 2012, 2014Jiang et al. 2017;Ma et al. 2018;Liu, Hu, et al. 2018;Liu, Tang, et al. 2018b), a chemical investigation on A. hongkongensis Hance was thus undertaken and had led to the isolation and characterization of a new naturally occurring trinoreudesmane sesquiterpene, artahongkongol A (1), together with seven known eudesmane sesquiterpenes. Their structures were elucidated on the basis of extensive spectroscopic methods. In addition, all isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines in vitro. Herein, we describe the isolation, structure elucidation, and cytotoxic properties of these compounds.

Results and discussion
The 90% EtOH extract of the stems and leaves of A. hongkongensis Hance was suspended in water and partitioned successively with petroleum ether and EtOAc. The petroleum ether fraction was repeatedly subjected to silica gel, Sephadex LH-20, reversed-phase C 18 silica gel column chromatography and semi-preparative HPLC, to yield eight eudesmane sesquiterpenes (1À8), including a new naturally occurring trinoreudesmane sesquiterpene, as shown in Figure 1.
Artahongkongol A (1) was obtained as white amorphous powder with a specific rotation of --57.2 (c 0.13, CH 3 OH). Its molecular formula, C 12 H 18 O 2 , was established by HRESIMS (m/z 195.1382 calcd 195.1380), indicating four degrees of unsaturation. Its IR spectrum showed the presence of a hydroxyl group (3398 cm À1 ) and an a,b-unsaturated ketone group (1683, 1608 and 1458 cm À1 ). The UV spectrum showed the absorption peaks at 209, 221 and 272 nm, which were characteristic of the a,b-unsaturated ketone. The 13 C NMR and DEPT data revealed the presence of 12 carbon atoms, including three sp 2 carbon atoms, one sp 3 quaternary carbon, two sp 3 methines, four sp 3 methines and two methyl groups. In addition, the three sp 2 carbon atoms were attributable to an a,b-unsaturated ketone group. The above data revealed that the structure of 1 was similar to that of 1a-hydroxy-5,11-eudesmadiene (2) (Nagashima et al. 2010), except the presence of the peak indicating the existence of a ketone carbonyl group of C-7 resonating at d C 200.0 in 1 and the absence of the peaks for the 2-propenyl group located at C-7 in 2, which was supported by the HMBC correlations of H-6, H-8a, H-8b and H-9a to C-7 (d C 200.0). Detailed analysis of 2D NMR (HSQC, HMBC, 1 H-1 H COSY and ROESY) spectra confirmed the planar structure of 1 as shown in Figure S1. The relative configurations of 1 were assigned on the basis of the ROESY correlations and the coupling constant of protons signals. In the ROESY spectrum of 1, the significant cross peaks between H 3 -14, H 3 -15 and H-2b indicated that 4-CH 3 and 10-CH 3 are located on the same side of the naphthalene ring, while the cross peaks between H-1, H-2a and H-3a suggested that H-1 is on the other side of the naphthalene ring ( Figure S1), which was further supported by the charactermatic coupling constant of H-1 (dd, J ¼ 11.8, 4.0 Hz), indicating the a-orientation of H-1 (Zhao et al. 1997;Guo et al. 1999;Nagashima et al. 2010). Thus, the structure of 1 was determined as shown in Figure 1.
All isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7 and SW480 using the MTT method, with doxorubicin as a positive control. It is noteworthy that compounds 1À8 showed significant cytotoxicities with IC 50 values ranging from 0.57 to 15.68 lM, which are similar to those of doxorubicin, even stronger inhibitory activities than that of doxorubicin in some cancer cell lines (see Table 1).

General experimental procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. IR spectra were obtained on a Nicolet 6700 spectrophotometer. NMR spectra were run on Bruker 400 MHz spectrometers using TMS as an internal standard. HRESIMS spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C 18 column (9.4 Â 250 mm, 5 mm). Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200-300 mesh) and octadecylsilyl silica gel (YMC; 50 lm) were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin layer chromatography (TLC).

Cytotoxicity bioassays
The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW480 (all cancer cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) in 5% CO 2 at 37 C. The cytotoxicity assay was performed using the MTT method in 96-well microplates (Jiang et al. 2017). Briefly, adherent cells (100 lL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, and suspended cells were seeded just before drug addition with an initial density of 1 Â 10 5 cells/mL. Each tumor cell line was exposed to the tested compound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 lM in triplicate for 48 h. Doxorubicin (Sigma, St. Louis, MO, USA) was used as a positive control. After treatment, cell viability was measured and the cell growth curve was plotted. IC 50 values were calculated by the Reed and Muench method (Jiang et al. 2017).

Conclusions
A new naturally occurring trinoreudesmane sesquiterpene, artahongkongol A (1), together with seven known eudesmane sesquiterpenes (2À8), was isolated from the stems and leaves of A. hongkongensis Hance. Among them, 1 is a rare trinoreudesmane sesquiterpene containing 12 carbon atoms on the carbon skeleton. All known compounds (2À8) were isolated from the genus Artabotrys for the first time. The discovery of 1 is not only a further addition to diverse and complex array of eudesmane sesquiterpenes, but also, the presence of 1À8 as characteristic marker may be helpful in chemotaxonomical classifications. Their cytotoxicities against several human cancer cell lines were also investigated, and found to be quite potent, which may be used as an explanation of the folk use of A. hongkongensis Hance, which had been used to treat tumors in China.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by the National Natural Science Foundation of China (Nos.